The Availability of Precision-Cut Lung Slices (PCLS) for Ex Vivo Studies

Respiratory diseases are considered one of the leading causes of death in humans. Chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IDF), asthma, and acute lung injury are among the leading respiratory disorders among humans. According to the American Lung Association, the number of people in the US suffering from COPD and related respiratory disorders is around 37 million. In fact, currently, there are roughly 1,400 people waiting to receive lung transplants, which is on the increase. The problem of pervasive pulmonary disorders in humans is compounded by the fact that no animal models can fully recapitulate human disorders. For this reason, developing new in vivo and ex vivo models to better understand the biological bases of pulmonary diseases and a mechanism to test novel therapeutic agents is in demand. Ex vivo models provide an opportunity to study both the functionality and structural characterization of samples. In this context, it has become obvious that developing ex vivo models for research on donated human lungs is well justified.

Donor Network West is in a unique position by i) having access to a large number of donated human lungs (~200/year), ii) the close proximity of the operation room to a wet laboratory, and iii) having access to all equipment and supplies to perform and optimize innovative tissue preservation methods.

Submit an application to request samples.

Soft tissues, like lungs, easily collapse during the transport and slicing. However, a major breakthrough called the precision-cut lung slices (PCLS) came by infusion of the airways of lungs with heated liquid agarose, which solidified at temperatures below 25 °C and therefore have the advantage to maintain the tissue structure. Among ex vivo models, PCLS technology offers many advantages including:

  1. The preservation of anatomy including alveolar architecture, respiratory parenchyma, structural and immune cell populations, and connective tissue.
  2. PCLS provides three-dimensional lung tissue slices that retain the majority of intact lung cells and their function.
  3. The slices containing cells are biologically active and the segments are similar to the condition found in vivo.
  4. Slices can be cryopreserved and used in culture studies from explanted human lungs particularly those with rare disorders.
  5. The slices are able to accurately reflect any changes to the underlying extracellular matrix associated with the disease.
  6. PCLS can be regarded as a “mini” lung that can be studied in the context of asthma, COPD, idiopathic pulmonary fibrosis, allergy, infections as well as in toxicology studies.
  7. PCLS has also been utilized to evaluate alternative targets for testing different therapeutic agents in addition to its use in toxicological studies.
  8. Frozen-thawed PCLSs show significant metabolic activity.

The tissue slices can be subsequently cultured with drug candidates or test substances. After several hours or days, the tissue response can be observed. For example, the airways in the tissue slices may show microscopic alterations, or release of signaling molecules can be found. In addition, they use this model to study the effects of new drugs – at an early stage of drug development.
Lung samples from research-consented donors with a number of pulmonary abnormalities can be collected, cut and frozen and shipped to the entire US.

Materials and Methods:

Agarose Filling:

Typically, agarose with a low-melting point is used in a concentration range of 0.5–3%. Once liquified at temperatures above 65 °C, agarose remains fluid when kept at 37 °C, solidifying only when the temperature drops below 25 °C.

PCLS Human Lung Slice Preparation:

The lungs are procured and perfused with 2 liters of saline or UW to remove the blood from the lungs. The lobe of interest is then filled with agarose. The solidified agarose maintains the inflated state of the lung and prevents the collapse of the airways and alveoli during slicing.

After the filling, the lung is immersed in an ice-cold buffer, initiating a rapid solidification of agarose, followed by precise cutting. In general, PCLS are prepared to a thickness of 100–500 μm. This process results in reproducible, uniform slices which can be deployed in a variety of ex vivo experimental protocols and therapeutics.

agarose filling lung

Following solidification of the lungs filled with heated agarose, multiple anatomically-defined segments are dissected.

Anatomically-defined lung segments will be sliced using a Vibratome. This device allows uniform slicing of lung segments into 100-1000 μm thick sections.

The prepared slices will be placed in an incubator. Typically, PCLSs are submerged in culture medium in multi-well plates at 37 °C, 5% CO2 and 95–100% air humidity under tissue culture conditions, and medium is changed daily. The culture medium has been optimized to be supplemented with essential nutrients, enabling culture of viable PCLS for up to 14 days, as compared to previous reports of tissue surviving for only 3–5 days. Furthermore, the addition of antibiotics such as penicillin and streptomycin prevent pathogen contamination from the onset of culture. Whilst in culture, PCLSs remained viable and maintained normal metabolic activity, tissue homeostasis, structural integrity, and responses to stimulation with lipopolysaccharide.

plcs cutting

Following incubation, the slices will be snap frozen and can be shipped across the United States.

In practice, PCLS can retain comparable viability and tissue homeostasis, either physiological or functional, during a cultivation period of 1 to 3 days, although extended periods can be achieved with optimized culture conditions. Another advantage of PCLS is the ability to store the generated slices by cryopreservation for future use, which is particularly critical for rare patient populations. PCLS can be cryopreserved at -80° C for a prolonged period of time. Following cryopreservation and thawing, PCLS retains vital functions of cells, enabling collection of many donors/phenotypes prior to experimentation.

Learn More:

For more information about acquiring human PCLS please contact Dr. Ahmad Salehi, [email protected].

Submit an application to request samples.

Learn more about additional research samples offered through Donor Network West’s Research Program.